Methods of treating an overweight or obese subject

ABSTRACT

The invention generally relates to methods of treating an overweight or obese subject, and treating overweight- or obesity-related conditions. In certain embodiments, the invention provides a method of treating an overweight or obese subject including administering a MetAP2 inhibitor in which the amount administered does not substantially modulate angiogenesis.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/133,060 filedOct. 27, 2011, which is a national stage filing under 35 U.S.C. §371 ofPCT/US2009/066816, filed Dec. 4, 2009, which claims priority to U.S.provisional applications U.S. Ser. No. 61/119,875 filed Dec. 4, 2008,U.S. Ser. No. 61/119,881 filed Dec. 4, 2008, U.S. Ser. No. 61/119,884filed Dec. 4, 2008, U.S. Ser. No. 61/119,886 filed Dec. 4, 2008, U.S.Ser. No. 61/119,872 filed Dec. 4, 2008, U.S. Ser. No. 61/119,877 filedDec. 4, 2008, U.S. Ser. No. 61/119,885 filed Dec. 4, 2008, U.S. Ser. No.61/119,891 filed Dec. 4, 2008, U.S. Ser. No. 61/119,888 filed Dec. 4,2008, U.S. Ser. No. 61/275,688 filed Aug. 3, 2009, and U.S. Ser. No.61/260,194 filed Nov. 11, 2009, each application of which is herebyincorporated by reference.

BACKGROUND

Obesity is a complex medical disorder of appetite regulation andmetabolism resulting in excessive accumulation of adipose tissue mass.Typically defined as a body mass index (BMI) of 30 kg/m² or more,obesity is a world-wide public health concern that is associated withcardiovascular disease, diabetes, certain cancers, respiratorycomplications, osteoarthritis, gallbladder disease, decreased lifeexpectancy, and work disability. The primary goals of obesity therapyare to reduce excess body weight, improve or prevent obesity-relatedmorbidity and mortality, and maintain long-term weight loss.

Treatment modalities typically include lifestyle management,pharmacotherapy, and surgery. Treatment decisions are made based onseverity of obesity, seriousness of associated medical conditions,patient risk status, and patient expectations. Notable improvements incardiovascular risk and the incidence of diabetes have been observedwith weight loss of 5-10% of body weight, supporting clinical guidelinesfor the treatment of obesity that recommend a target threshold of 10%reduction in body weight from baseline values.

However, while prescription anti-obesity medications are typicallyconsidered for selected patients at increased medical risk because oftheir weight and for whom lifestyle modifications (diet restriction,physical activity, and behavior therapy) alone have failed to producedurable weight loss, approved drugs have had unsatisfactory efficacy forseverely obese subjects, leading to only ˜3-5% reduction in body weightafter a year of treatment.

Bariatric surgery may be considered as a weight loss intervention forpatients at or exceeding a BMI of 40 kg/m². Patients with a BMI≧35 kg/m²and an associated serious medical condition are also candidates for thistreatment option. Unfortunately, postoperative complications commonlyresult from bariatric surgical procedures, including bleeding, embolismor thrombosis, wound complications, deep infections, pulmonarycomplications, and gastrointestinal obstruction; reoperation during thepostoperative period is sometimes necessary to address thesecomplications. Rates of reoperation or conversion surgery beyond thepostoperative period depend on the type of bariatric procedure, and inone study ranged from 17% to 31%. Intestinal absorptive abnormalities,such as micronutrient deficiency and protein-calorie malnutrition, alsoare typically seen with bypass procedures, requiring lifelong nutrientsupplementation. Major and serious adverse outcomes associated withbariatric surgery are common, observed in approximately 4 percent ofprocedures performed (including death in 0.3 to 2 percent of allpatients receiving laparoscopic banding or bypass surgeries,respectively)

MetAP2 encodes a protein that functions at least in part byenzymatically removing the amino terminal methionine residue fromcertain newly translated proteins such as glyceraldehyde-3-phosphatedehydrogenase (Warder et al. (2008) J Proteome Res 7:4807). Increasedexpression of the MetAP2 gene has been historically associated withvarious forms of cancer. Molecules inhibiting the enzymatic activity ofMetAP2 have been identified and have been explored for their utility inthe treatment of various tumor types (Wang et al. (2003) Cancer Res.63:7861) and infectious diseases such as microsporidiosis,leishmaniasis, and malaria (Zhang et al. (2002) J. Biomed. Sci. 9:34).However, such MetAP2 inhibitors may be useful as well for patients withexcess adiposity and conditions related to adiposity including type 2diabetes, hepatic steatosis, and cardiovascular disease (via e.g.ameliorating insulin resistance, reducing hepatic lipid content, andreducing cardiac workload). Methods of treating obese subjects that aremore effective than e.g. dieting alone are clearly needed.

SUMMARY

This disclosure generally relates to methods of treating an overweightor obese subject or patient that include non-parenterally administeringa pharmaceutically effective amount of a MetAP2 inhibitor to a patientin need thereof, e.g., a human or a companion animal such as a cat or adog.

In one embodiment, a method of treating obesity in a patient in needthereof is provided, comprising non-parenterally administering apharmaceutically effective amount of a MetAP2 inhibitor to said patient.Such methods may result in, for example, a lower systemic exposure tosaid MetAP2 inhibitor as compared to a patient parenterally administeredthe same of amount of the MetAP2 inhibitor. Contemplatedpharmaceutically effective amounts may not substantially modulate orsuppress angiogenesis. In exemplary embodiments, non-parenteraladministration may result in decreased body fat and a substantialmaintenance of muscle mass in said patient. Such methods may enhance fatoxidation compared to a patient on a restricted food intake diet alone.Also provided herein is a method of treating obesity in a patient inneed thereof, comprising administering a pharmaceutically effectiveamount of a MetAP2 inhibitor to said patient, wherein substantially noloss of new blood vessels in fat deposits occur as compared to a patientbeing treated for obesity using an energy restricted diet alone. In someembodiments, non-parenterally administration may include oral, buccal,sublingual, transdermal, rectal, nasal administration, or administrationvia inhalation.

Contemplated MetAP2 inhibitors include substantially irreversibleinhibitors, such as e.g., fumagillin, fumagillol or fumagillin ketonederivative, siRNA, shRNA, an antibody, or a antisense compound, or,e.g., O-(4-dimethylaminoethoxycinnamoyl)fumagillol and pharmaceuticallyacceptable salts thereof In another embodiment, a contemplated MetAP2inhibitor may be substantially reversible inhibitor.

Also provided herein is a method for controlling or preventing hepaticsteatosis in an obese patient being treated for obesity, comprisingadministering a pharmaceutically effective amount of a MetAP2 inhibitorto said patient, and/or a method for improving liver function in anobese patient, comprising administering a pharmaceutically effectiveamount of a MetAP2 inhibitor to said patient.

In an embodiment, a method of improving exercise capacity in a patientin need thereof is provided that includes administering apharmaceutically effective amount of a MetAP2 inhibitor to said patient.

A method of reducing weight of a patient in a patient in need thereof iscontemplated herein that comprises administering a pharmaceuticallyeffective amount of a MetAP2 inhibitor to said patient wherein themetabolic rate of the patient is not substantially reduced as comparedto the metabolic rate of a diet only patient on an energy restricteddiet alone. Also provided herein is a method of restoring normalmetabolic action in an obese patient in need thereof, comprisingadministering a pharmaceutically effective amount of a MetAP2 inhibitorto said patient.

In an embodiment, a method of decreasing body fat in an overweight orobese patient in need thereof is provided that comprises administering apharmaceutically effective amount of a MetAP2 inhibitor to said patientresulting in body fat reduction, and wherein said patient substantiallymaintains muscle mass during the body fat reduction, for example apatient may retain substantially more muscle mass as compared to bodyfat reduction in a patient using an energy restricted diet alone.

A method of activating brown fat function and/or increasing brown fattissue mass in a patient in need thereof is provided, comprisingadministering a pharmaceutically effective amount of a MetAP2 inhibitorto said patient, and/or a method of restoring and/or maintaining thyroidhormone concentrations in an obese patient, comprising administering apharmaceutically effective amount of a MetAP2 inhibitor to said patient.

The disclosed methods may include a pharmaceutically effective amount ofa MetAP2 inhibitor that does not substantially modulate or suppressangiogenesis in a treated patient. In some embodiments of the disclosedmethods, a patient has a lower systemic exposure to said MetAP2inhibitor as compared to a patient parenterally administered the sameamount of the MetAP2 inhibitor.

MetAP2 inhibitors may be administered non-parenterally, orally. buccallyor sublingually, topically, rectally, or transdermally in at least somedisclosed methods. In some disclosed methods, a MetAP2 inhibitor may beadministered subcutaneously or intravenously. In some embodiments,administration of a MetAP2 inhibitor may occur at least daily, or everyother day, or at least weekly.

Also contemplated herein is a method for reducing the amount orfrequency of administering supplemental insulin in a patient sufferingfrom type 2 diabetes, comprising a pharmaceutically effective amount ofa MetAP2 inhibitor.

A method for improving surgical outcome in an obese patient in needthereof, by reducing weight of said patient is provided herein,comprising administering a pharmaceutically effective amount of a MetAP2inhibitor to said patient before non-acute surgery, thereby reducingliver and/or abdominal fat in said patient and improving surgicaloutcome. Such surgeries may include e.g., bariatric surgery,cardiovascular surgery, abdominal surgery, or orthopedic surgery. Alsoprovided herein is a method of maintaining a specified weight in aformerly obese patient, comprising administering a pharmaceuticallyeffective amount of a MetAP2 inhibitor to said patient.

Disclosed methods may further comprise co-administering an additionalweight loss agent and/or may further comprise administering a foodrestricted diet to a patient. In another embodiment, a disclosed methodmay further comprise assessing the ketone body production level in apatient; and optionally adjusting the amount administered; therebyoptimizing the therapeutic efficacy of said MetAP2 inhibitor. In atleast some disclosed methods, a patient may incur greater than or aboutequal to a 20% weight loss after about 6 months of said administration.

In another embodiment, a method for treating obesity in a patient inneed thereof is provided, comprising administering about 0.005 to about0.04 mg/kg of a MetAP2 inhibitor selected fromO-(4-dimethylaminoethoxycinnamoyl)fumagillol and pharmaceuticallyacceptable salts thereof, for example, an oxalate salt, to said patient.This MetAP2 inhibitor may be administered at least daily, or 1, 2, 3 or4 times a week. In some embodiments, MetAP2 may be administeredparenterally, e.g., intravenously, or non-parenterally. Uponadministration of the MetAP2 inhibitor e.g. daily, or 1, 2, 3, 4, 5, 6or 7 times a week, for about 6 months, may result in at least a 20%weight loss or more of the patient's original weight.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a bar graph showing TXN(104) in B16F10 cell lysate afterchymotryptic digest.

FIG. 2 is a bar graph showing N-acetyl TXN(1-4) in B16F10 cell lysateafter chymotriptic digest.

FIG. 3 is a photograph of a gel showing that fumagillin significantlyincreased TXN in both Cos-1 and 293T cells.

FIG. 4 is a photograph of a gel showing that EC50 of fumagillin toincrease TXN levels is <10 nM.

FIG. 5A is a bar graph showing the concentration of beta-hydroxybutyratein mice fed a high fat diet (vehicle) or mice fed a high-fat diet plusoral administration of fumagillin for ten days at the concentrationsindicated. FIG. 5B is a bar graph showing the concentration ofnonesterified fatty acid concentrations under the conditions describedfor FIG. 5A. FIG. 5C is a bar graph showing the concentration ofbeta-hydroxybutyrate in mice fed a high fat diet (vehicle) or mice fed ahigh-fat diet plus oral administration of fumagillin for 250 days at theconcentrations indicated.

FIG. 6 is a line graph showing the body weight over time of high-fatdiet-fed C57BL/6 mice treated with 1 mg/kg fumagillin (squares) orvehicle (diamonds) for 250 days. Age-matched lean C57BL/6 micemaintained on normal mouse chow diet are shown for comparison(triangles).

FIG. 7 shows age-matched lean mice maintained on normal mouse chow diet(left) or mice fed a high fat diet-fed and treated with 1 mg/kgfumagillin (right) or vehicle (middle) for 250 days.

FIG. 8 contains two bar graphs showing a comparison of retroperitonealfat pad weight in grams (left graph) and as a percentage of total bodyweight (right graph) for mice fed a high-fat diet (diet-induced obesity(DIO), left bar), mice fed a high-fat diet plus 1 mg/kg fumagillin(DIO+ZGN-201, middle bar), and age-matched lean mice (right bar).

FIG. 9 contains three bar graphs showing concentrations of glucose (topleft graph), insulin (top right graph) and glucose-insulin complex(bottom graph) in mice fed a high-fat diet (diet-induced obesity (DIO),left bar), mice fed a high-fat diet plus 1 mg/kg fumagillin(DIO+ZGN-201, middle bar), and age-matched lean mice (right bar).

FIG. 10 shows two line graphs displaying body weight (left graph) andfood intake (right graph) of mice fed a high fat diet-fed C57BL/6 miceand treated with 1 mg/kg of the MetAP2 inhibitor fumagillin (squares) orvehicle (diamonds) for 14 days.

FIG. 11 is a bar graph showing the percentage change in fat tissue andlean tissue in mice conditioned on a high fat (HF) diet, miceconditioned on a high-fat diet and treated with fumagillin (ZGN-201)administered by oral gavage at doses of 0.1 or 0.3 mg/kg daily, or micefed on a low-fat diet for 28 days. For each of “Fat Tissue Change” and“Lean Tissue Change,” bars from left to right are: LF, HF, HF+0.3, andHF+0.1.

FIG. 12 contains two histological images showing a comparison ofretroperitoneal fat pad brown adipose tissue content for mice fed ahigh-fat diet (diet-induced obesity (DIO)) (left image), and mice fed ahigh-fat diet plus 1 mg/kg fumagillin (DIO+ZGN-201 (right image).

FIG. 13 depicts the effect of 7 day administration of various MetAP2inhibitors on body weight in DIO mice.

DETAILED DESCRIPTION

Overview

Obesity and being overweight refer to an excess of fat in proportion tolean body mass. Excess fat accumulation is associated with increase insize (hypertrophy) as well as number (hyperplasia) of adipose tissuecells. Obesity is variously measured in terms of absolute weight,weight:height ratio, degree of excess body fat, distribution ofsubcutaneous fat, and societal and esthetic norms. A common measure ofbody fat is Body Mass Index (BMI). The BMI refers to the ratio of bodyweight (expressed in kilograms) to the square of height (expressed inmeters). Body mass index may be accurately calculated using theformulas: SI units: BMI=weight(kg)/(height²(m²), or US units:BMI=(weight(lb)*703)/(height²(in²).

In accordance with the U.S. Centers for Disease Control and Prevention(CDC), an overweight adult has a BMI of 25 kg/m² to 29.9 kg/m², and anobese adult has a BMI of 30 kg/m² or greater. A BMI of 40 kg/m² orgreater is indicative of morbid obesity or extreme obesity. Forchildren, the definitions of overweight and obese take into account ageand gender effects on body fat.

BMI does not account for the fact that excess adipose can occurselectively in different parts of the body, and development of adiposetissue can be more dangerous to health in some parts of the body ratherthan in other parts of the body. For example, “central obesity”,typically associated with an “apple-shaped” body, results from excessadiposity especially in the abdominal region, including belly fat andvisceral fat, and carries higher risk of co-morbidity than “peripheralobesity”, which is typically associated with a “pear-shaped” bodyresulting from excess adiposity especially on the hips. Measurement ofwaist/hip circumference ratio (WHR) can be used as an indicator ofcentral obesity. A minimum WHR indicative of central obesity has beenvariously set, and a centrally obese adult typically has a WHR of about0.85 or greater if female and about 0.9 or greater if male.

Methods of determining whether a subject is overweight or obese thataccount for the ratio of excess adipose tissue to lean body mass mayinvolve obtaining a body composition of the subject. Body compositioncan be obtained by measuring the thickness of subcutaneous fat inmultiple places on the body, such as the abdominal area, the subscapularregion, arms, buttocks and thighs. These measurements are then used toestimate total body fat with a margin of error of approximately fourpercentage points. Another method is bioelectrical impedance analysis(BIA), which uses the resistance of electrical flow through the body toestimate body fat. Another method is using a large tank of water tomeasure body buoyancy. Increased body fat will result in greaterbuoyancy, while greater muscle mass will result in a tendency to sink.Another method is fan-beam dual energy X-ray absorptiometry (DEXA). DEXAallows body composition, particularly total body fat and/or regional fatmass, to be determined non-invasively.

Without being limited by any particular theory or mechanism of action,it is believed that fat oxidation and lipolysis are stimulated throughtreatment with inhibitors of MetAP2 that enhance the level and functionof thioredoxin and/or over-rides the inhibitory effects ofhyperinsulinemia related at least in part to insulin-stimulation and/orover-rides the inhibitory effects of high fat diet induced NADPH oxidaseactivity. A coordinated action can be induced which leads to aphysiological reduction in body adiposity through increased loss of fattissue-associated triglyceride, enhanced local generation of3,5,3′-triiodothyronine active thyroid hormone with correspondingenhanced activity of brown adipose tissue and its sensitivity tophysiological stimuli, increased metabolism of free fatty acids by theliver with increased ketone body formation, and reduced food intake.These effects are evident at doses of a MetAP2 inhibitor that do notsubstantially modulate angiogenesis.

In obese and/or hyperinsulinemic patients, liver PKA function may besuppressed secondary to elevated NADPH oxidase expression. Ketone bodyproduction and utilization are typically suppressed in an obese patient,potentially reducing hepatic satiety signals and increasing foodconsumption. However, administration of a MetAP2 inhibitor, withoutbeing limited by an theory, leads to inhibition of thioredoxinamino-terminal methionine processing and increases steady-statethioredoxin levels, reactivating protein kinase A (PKA) function,reactivating adipose tissue lipase activity and/or stimulatingproduction and/or activity of the rate-limiting enzyme ofbeta-hydroxybutyrate production (3-hydroxymethyl glutaryl CoA synthase),leading to elevated ketone body production, as illustrated in e.g. FIG.5.

The coordinated and physiologic induction of anti-obesity activitiesmediated by the methods of the present invention may lead to a healthyreduction in tissue levels of triglyceride, diacylglycerol, and otherfat-related mediators and oxidants, and can result in a new steady statesituation that favors lean body composition and increased whole bodyenergy metabolism. Without being bound by any theory, it is believedthat the mechanistic cascade activated by MetAP2 inhibitors leads to fattissue being converted to ketone bodies and burned as fuel, unlikeexisting therapies (including e.g., calorie or energy restricted diets)that target central control of food intake and that may carry adverseside effects (e.g. adverse neurological side effects). Further,therapeutically effective doses contemplated herein will not typicallyinduce any anti-angiogenic action.

MetAP2 Inhibitors

MetAP2 inhibitors refer to a class of molecules that inhibit theactivity of MetAP2, e.g., the ability of MetAP2 to cleave the N-terminalmethionine residue of newly synthesized proteins to produce the activeform of the protein, or the ability of MetAP2 to regulate proteinsynthesis by protecting the subunit of eukaryotic initiation factor-2(eIF2) from phosphorylation.

Exemplary MetAP2 inhibitors may include irreversible inhibitors thatcovalently bind to MetAP2. For example, such irreversible inhibitorsinclude fumagillin, fumagillol, fumagillin ketone, Fumagillin refers toall stereoisomers, and can be represented by the following structure:

Fumagillol refers to all stereoisomers of the following structure:

Fumagillin ketone refers to all stereoisomers of the followingstructure, and can be represented by the following structure:

Derivatives and analogs of fumagillin, and pharmaceutically acceptablesalts thereof are contemplated herein as irreversible MetAP2 inhibitors,such as O-(4-dimethylaminoethoxycinnamoyl)fumagillol (CKD-732, alsoreferred to herein as Compound A),O-(3,4,5-trimethoxycinnamoyl)fumagillol,O-(4-chlorocinnamoyl)fumagillol; O-(4-aminocinnamoyl)fumagillol;O-(4-dimethylaminoethoxycinnamoyl)fumagillol;O-(4-methoxycinnamoyl)fumagillol;O-(4-dimethylaminocinnamoyl)fumagillol;O-(4-hydroxycinnamoyl)fumagillol; O-(3,4-dimethoxycinnamoyl)fumagillol;O-(3,4-methylenedioxycinnamoyl)fumagillol;O-(3,4,5-trimethoxycinnamoyl)fumagillol; O-(4-nitrocinnamoyl)fumagillol;O-(3,4-dimethoxy-6-aminocinnamoyl)fumagillol;O-(4-acetoxy-3,5-dimethoxycinnamoyl)fumagillol;O-(4-ethylaminocinnamoyl)fumagillol;O-(4-ethylaminoethoxycinnamoyl)fumagillol;O-(3-dimethylaminomethyl-4-methoxycinnamoyl)fumagillol;O-(4-trifluoromethylcinnamoyl)fumagillol;O-(3,4-dimethoxy-6-nitrocinnamoyl)fumagillol;O-(4-acetoxycinnamoyl)fumagillol; O-(4-cyanocinnamoyl)fumagillol;4-(4-methoxycinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol;O-(3,4,5-trimethoxycinnamoyl)fumagillol;O-(4-dimethylaminocinnamoyl)fumagillol;O-(3,4,5-trimethoxycinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-m-ethoxy-1-chloromethyl-1-cyclohexanol;O-(4-dimethylaminocinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-me-thoxy-1-chloromethyl-1-cyclohexanol;O-(3,5-dimethoxy-4-hydroxycinnamoyl)fumagillol orO-(chloracetyl-carbamoyl)fumagillol(TNP-470).

Fumagillin, and some derivatives thereof, have a carboxylic acid moietyand can be administered in the form of the free acid. Alternatively,contemplated herein are pharmaceutically acceptable salts of fumagillin,fumagillol, and derivatives thereof. Pharmaceutically acceptable saltsillustratively include those that can be made using the following bases:ammonia, L-arginine, benethamine, benzathene, betaine, bismuth, calciumhydroxide, choline, deanol, diethanolamine, diethylarnine,2-(diethylamino)ethanol, ethylenediamine, N-methylglucarnine,hydrabamine, 1H-imidazole, lysine, magnesium hydroxide,4-(2-hydroxyethyl)morpholine, piperazine, potassium hydroxide,1-(2-hydroxyethyl)pyrrolidine, sodium hydroxide, triethanolamine, zinchydroxide, diclyclohexlamine, or any other electron pair donor (asdescribed in Handbook of Pharmaceutical Salts, Stan & Wermuth, VHCA andWiley, Uchsenfurt-Hohestadt Germany, 2002). Contemplatedpharmaceutically acceptable salts may include hydrochloric acid, bromicacid, sulfuric acid, phosphoric acid, nitric acid, formic acid, aceticacid, trifluoroacetic acid, oxalic acid, fumaric acid, tartaric acid,maleic acid, methanesulfonic acid, benzenesulfonic acid orpara-toluenesulfonic acid.

Esters of the present invention may be prepared by reacting fumagillinor fumagillol with the appropriate acid under standard esterificationconditions described in the literature (Houben-Weyl 4th Ed. 1952,Methods of Organic Synthesis). Suitable fumagillin esters include ethylmethanoate, ethyl ethanoate, ethyl propanoate, propyl methanoate, propylethanoate, and methyl butanoate.

In another embodiment, contemplated irreversible inhibitors of MetAP2may include a siRNA, shRNA, an antibody or an antisense compound ofMetAP2.

Further examples of MetAP2 inhibitors, are provided in the followingreferences, each of which is hereby incorporated by reference: Olson etal. (U.S. Pat. No. 7,084,108 and WO 2002/042295), Olson et al. (U.S.Pat. No. 6,548,477; U.S. Pat. No. 7,037,890; U.S. Pat. No. 7,084,108;U.S. Pat. No. 7,268,111; and WO 2002/042295), Olson et al. (WO2005/066197), Hong et al. (U.S. Pat. No. 6,040,337), Hong et al. (U.S.Pat. No. 6,063,812 and WO 1999/059986), Lee et al. (WO 2006/080591),Kishimoto et al. (U.S. Pat. No. 5,166,172; U.S. Pat. No. 5,698,586; U.S.Pat. No. 5,164,410; and 5,180,738), Kishimoto et al. (U.S. Pat. No.5,180,735), Kishimoto et al. (U.S. Pat. No. 5,288,722), Kishimoto et al.(U.S. Pat. No. 5,204,345), Kishimoto et al. (U.S. Pat. No. 5,422,363),Liu et al. (U.S. Pat. No. 6,207,704; U.S. Pat. No. 6,566,541; and WO1998/056372), Craig et al. (WO 1999/057097), Craig et al. (U.S. Pat. No.6,242,494), BaMaung et al. (U.S. Pat. No. 7,030,262), Comess et al. (WO2004/033419), Comess et al. (US 2004/0157836), Comess et al. (US2004/0167128), Henkin et al. (WO 2002/083065), Craig et al. (U.S. Pat.No. 6,887,863), Craig et al. (US 2002/0002152), Sheppard et al. (2004,Bioorganic & Medicinal Chemistry Letters 14:865-868), Wang et al. (2003,Cancer Research 63:7861-7869), Wang et al. (2007, Bioorganic & MedicinalChemistry Letters 17:2817-2822), Kawai et al. (2006, Bioorganic &Medicinal Chemistry Letters 16:3574-3577), Henkin et al. (WO2002/026782), Nan et al. (US 2005/0113420), Luo et al. (2003, J. Med.Chem., 46:2632-2640), Vedantham et al. (2008, J. Comb. Chem.,10:195-203), Wang et al. (2008, J. Med. Chem., XXXX, Vol. xxx, No. xx),Ma et al. (2007, BMC Structural Biology, 7:84) and Huang et al. (2007,J. Med. Chem., 50:5735-5742), Evdokimov et al. (2007, PROTEINS:Structure, Function, and Bioinformatics, 66:538-546), Garrabrant et al.(2004, Angiogenesis 7:91-96), Kim et al. (2004, Cancer Research,64:2984-2987), Towbin et al. (2003, The Journal of Biological Chemistry,278(52):52964-52971), Marino Jr. (U.S. Pat. No. 7,304,082), Kallender etal. (U.S. patent application number 2004/0192914), and Kallender et al.(U.S. patent application numbers 2003/0220371 and 2005/0004116). In someembodiments, contemplated MetAP2 inhibitors do not include fumagillin,fumagillol, fumagillin ketone, CKD-732/Compound A, and/or TNP-470.

For example, contemplated MetAP2 inhibitors may include:

Methods

A method of treating obesity in a patient in need thereof is providedherein, comprising non-parenterally administering a pharmaceuticallyeffective amount of a MetAP2 inhibitor to said patient. In someembodiments, a contemplated pharmaceutically effective amount of aMetAP2 as described below, does not substantially modulate or suppressangiogenesis, but is still effective as MetAP2 inhibitor. The term“angiogenesis” is known to persons skilled in the art, and refers to theprocess of new blood vessel formation, and is essential for theexponential growth of solid tumors and tumor metastasis. For example,provided herein is a method of treating obesity in a patient in needthereof, comprising administering a pharmaceutically effective amount ofa MetAP2 inhibitor to said patient, wherein substantially no loss of newblood vessels in fat deposits or other tissue compartments occur ascompared to a patient being treated for obesity using an energyrestricted diet alone.

For example, fumagillin-class molecules irreversibly inhibit enzymaticactivity of MetAP2, leading to N-terminal acetylation and stabilizationof these proteins at doses considerably lower than those required tosuppress angiogenesis or tumor growth in vivo. Without being limited toany theory, the long-lasting covalent inhibition of MetAP2 enzymaticactivity driven by such MetAP2 inhibitors may be responsible for thesegregation of angiogenic effects from metabolic responses mediated byincreased thioredoxin and/or glyceraldehyde-3-phosphate levels in vivo.Alternatively, anti-tumor effects driven by angiogenesis inhibition mayrequire a more thorough starvation of the tumor by heavily restrictingblood supply, which requires high doses. Metabolic effects, however, mayrequire a minor and incomplete perturbation of the system which occursat lower doses and without any obvious direct effect on blood vessels.

Treated patients used the disclosed methods may have a lower systemicexposure to said MetAP2 inhibitor as compared to a patient parenterallyadministered the same of amount of the MetAP2 inhibitor. In an exemplaryembodiment, the disclosed methods may result in less accumulation in thereproductive tract (e.g. testis) of a patient, for example, as comparedto the same amount of MetAP2 inhibitor subcutaneously administered.

Disclosed methods of treating obesity e.g by non-parenterallyadministering a MetAP2 inhibitor, may result in decreased body fat and asubstantial maintenance of muscle mass in said patient. In certainembodiments, upon administration, fat oxidation is enhanced in a patientas compared to a patient on a restricted food intake diet alone. Forexample, provided herein is a method of decreasing body fat in anoverweight or obese patient in need thereof, comprising administering apharmaceutically effective amount of a MetAP2 inhibitor to said patientresulting in body fat reduction, and wherein said patient substantiallymaintains muscle mass during the body fat reduction. Such a patient mayretain substantially more muscle mass as compared to body fat reductionin a patient using an energy restricted diet alone.

In some embodiments, disclosed methods, upon administration of saidMetAP2 inhibitor e.g. daily or weekly, for about 3, 4, 5 or 6 months ormore may result in at least a 5%, 10%, 20%, or 30%, or more weight lossbased on the patient's original weight. In an embodiment, weight lossfollowing treatment with therapeutically effective doses of MetAP2inhibitors may substantially cease once a patient attains a normal bodycomposition. Without being limited to an theory, this may be due toreliance of the mechanism on re-establishing tone of adrenergic signaltransduction in tissues such as fat, liver, and/or skeletal muscle.

In an embodiment, provided herein is a method of maintaining a specifiedweight in a formerly obese patient, comprising administering apharmaceutically effective amount of a MetAP2 inhibitor to said patient.

Also provided herein is a method for controlling or preventing hepaticsteatosis in an obese patient being treated for obesity, comprisingadministering a pharmaceutically effective amount of a MetAP2 inhibitorto said patient. In another embodiment, a method for improving liverfunction in an obese patient is provided, comprising administering apharmaceutically effective amount of a MetAP2 inhibitor to said patient.For example, a method of restoring normal metabolic action in an obesepatient in need thereof is provided, comprising administering apharmaceutically effective amount of a MetAP2 inhibitor to said patient.In an embodiment, a method of reducing weight of a patient in a patientin need thereof is provided comprising administering a pharmaceuticallyeffective amount of a MetAP2 inhibitor to said patient wherein themetabolic rate of the patient is not substantially reduced as comparedto the metabolic rate of a diet only patient on an energy restricteddiet alone. In a different embodiment, a method of restoring and/ormaintaining thyroid hormone concentrations in an obese patient isprovided, comprising administering a pharmaceutically effective amountof a MetAP2 inhibitor to said patient.

In an embodiment, a method of improving exercise capacity in a patientin need thereof is provided that comprises administering apharmaceutically effective amount of a MetAP2 inhibitor to said patient.

Also provided herein is a method of activating brown fat function in apatient in need thereof, comprising administering a pharmaceuticallyeffective amount of a MetAP2 inhibitor to said patient.

Contemplated herein is a method of reducing the amount or frequency ofadministering supplemental insulin in a patient suffering from type 2diabetes, comprising administering a pharmaceutically effective amountof a MetAP2 inhibitor to said patient. Such treatment may be directed toan obese or non-obese patient.

In an embodiment, a method for improving surgical outcome in an obesepatient in need thereof by reducing weight of said patient is providedcomprising administering a pharmaceutically effective amount of a MetAP2inhibitor to said patient before non-acute surgery, thereby reducingliver and/or abdominal fat in said patient and improving surgicaloutcome. Such surgeries may include bariatric surgery, cardiovascularsurgery, abdominal surgery, or orthopedic surgery.

In addition to being overweight or obese, a subject can further have anoverweight- or obesity-related co-morbidities, i.e., diseases and otheradverse health conditions associated with, exacerbated by, orprecipitated by being overweight or obese. Because being overweight orobese is associated with other adverse health conditions orco-morbidities, for example diabetes, administering MetAP2 inhibitorsbrings a benefit in ameliorating, arresting development of or, in somecases, even eliminating, these overweight- or obesity-related conditionsor co-morbidities. In some embodiments, methods provided herein mayfurther include administering at least one other agent that is directedto treatment of these overweight- or obesity-related conditions.

Contemplated other agents include those administered to treat type 2diabetes such as sulfonylureas (e.g., chlorpropamide, glipizide,glyburide, glimepiride); meglitinides (e.g., repaglinide andnateglinide); biguanides (e.g., metformin); thiazolidinediones(rosiglitazone, troglitazone, and pioglitazone); glucagon-like 1 peptidemimetics (e.g. exenatide and liraglutide); sodium-glucose cotransporterinhibitors (e.g., dapagliflozin), renin inhibitors, andalpha-glucosidase inhibitors (e.g., acarbose and meglitol), and/or thoseadministered to treat cardiac disorders and conditions, suchhypertension, dyslipidemia, ischemic heart disease, cardiomyopathy,cardiac infarction, stroke, venous thromboembolic disease and pulmonaryhypertension, which have been linked to overweight or obesity, forexample, chlorthalidone; hydrochlorothiazide; indapamide, metolazone;loop diuretics (e.g., bumetanide, ethacrynic acid, furosemide, lasix,torsemide); potassium-sparing agents (e.g., amiloride hydrochloride,spironolactone, and triamterene); peripheral agents (e.g., reserpine);central alpha-agonists (e.g., clonidine hydrochloride, guanabenzacetate, guanfacine hydrochloride, and methyldopa); alpha-blockers(e.g., doxazosin mesylate, prazosin hydrochloride, and terazosinhydrochloride); beta-blockers (e.g., acebutolol, atenolol, betaxolol,nisoprolol fumarate, carteolol hydrochloride, metoprolol tartrate,metoprolol succinate, Nadolol, penbutolol sulfate, pindolol, propranololhydrochloride, and timolol maleate); combined alpha- and beta-blockers(e.g., carvedilol and labetalol hydrochloride); direct vasodilators(e.g., hydralazine hydrochloride and minoxidil); calcium antagonists(e.g., diltiazem hydrochloride and verapamil hydrochloride);dihydropyridines (e.g., amlodipine besylate, felodipine, isradipine,nicardipine, nifedipine, and nisoldipine); ACE inhibitors (benazeprilhydrochloride, captopril, enalapril maleate, fosinopril sodium,lisinopril, moexipril, quinapril hydrochloride, ramipril, trandolapril);angiotensin II receptor blockers (e.g., losartan potassium, valsartan,and Irbesartan); and combinations thereof, as well as statins such asmevastatin, lovastatin, pravastatin, simvastatin, velostatin,dihydrocompactin, fluvastatin, atorvastatin, dalvastatin, carvastatin,crilvastatin, bevastatin, cefvastatin, rosuvastatin, pitavastatin, andglenvastatin., typically for treatment of dyslipidemia.

Other agents that may be co-administered (e.g. sequentially orsimultaneously) include agents administered to treat ischemic heartdisease including statins, nitrates (e.g., Isosorbide Dinitrate andIsosorbide Mononitrate), beta-blockers, and calcium channel antagonists,agents administered to treat cardiomyopathy including inotropic agents(e.g., Digoxin), diuretics (e.g., Furosemide), ACE inhibitors, calciumantagonists, anti-arrhythmic agents (e.g., Sotolol, Amiodarone andDisopyramide), and beta-blockers, agents administered to treat cardiacinfarction including ACE inhibitors, Angiotensin II receptor blockers,direct vasodilators, beta blockers, anti-arrhythmic agents andthrombolytic agents (e.g., Alteplase, Retaplase, Tenecteplase,Anistreplase, and Urokinase), agents administered to treat strokesincluding anti-platelet agents (e.g., Aspirin, Clopidogrel,Dipyridamole, and Ticlopidine), anticoagulant agents (e.g., Heparin),and thrombolytic agents, agents administered to treat venousthromboembolic disease including anti-platelet agents, anticoagulantagents, and thrombolytic agents, agents administered to treat pulmonaryhypertension include inotropic agents, anticoagulant agents, diuretics,potassium (e.g., K-dur), vasodilators (e.g., Nifedipine and Diltiazem),Bosentan, Epoprostenol, and Sildenafil, agents administered to treatasthma include bronchodilators, anti-inflammatory agents, leukotrieneblockers, and anti-Ige agents. Particular asthma agents includeZafirlukast, Flunisolide, Triamcinolone, Beclomethasone, Terbutaline,Fluticasone, Formoterol, Beclomethasone, Salmeterol, Theophylline, andXopenex, agents administered to treat sleep apnea include Modafinil andamphetamines, agents administered to treat nonalcoholic fatty liverdisease include antioxidants (e.g., Vitamins E and C), insulinsensitizers (Metformin, Pioglitazone, Rosiglitazone, and Betaine),hepatoprotectants, and lipid-lowering agents, agentsadministered totreat osteoarthritis of weight-bearing joints include Acetaminophen,non-steroidal anti-inflammatory agents (e.g., Ibuprofen, Etodolac,Oxaprozin, Naproxen, Diclofenac, and Nabumetone), COX-2 inhibitors(e.g., Celecoxib), steroids, supplements (e.g. glucosamine andchondroitin sulfate), and artificial joint fluid, agents administered totreat Prader-Willi Syndrome include human growth hormone (HGH),somatropin, and weight loss agents (e.g., Orlistat, Sibutramine,Methamphetamine, Ionamin, Phentermine, Bupropion, Diethylpropion,Phendimetrazine, Benzphetermine, and Topamax), agents administered totreat polycystic ovary syndrome include insulin-sensitizers,combinations of synthetic estrogen and progesterone, Spironolactone,Eflornithine, and Clomiphene, agents administered to treat erectiledysfunction include phosphodiesterase inhibitors (e.g., Tadalafil,Sildenafil citrate, and Vardenafil), prostaglandin E analogs (e.g.,Alprostadil), alkaloids (e.g., Yohimbine), and testosterone, agentsadministered to treat infertility include Clomiphene, Clomiphenecitrate, Bromocriptine, Gonadotropin-releasing Hormone (GnRH), GnRHagonist, GnRH antagonist, Tamoxifen/nolvadex, gonadotropins, HumanChorionic Gonadotropin (HCG), Human Menopausal Gonadotropin (HmG),progesterone, recombinant follicle stimulating hormone (FSH),Urofollitropin, Heparin, Follitropin alfa, and Follitropin beta, agentsadministered to treat obstetric complications include Bupivacainehydrochloride, Dinoprostone PGE2, Meperidine HCl,Ferro-folic-500/iberet-folic-500, Meperidine, Methylergonovine maleate,Ropivacaine HCl, Nalbuphine HCl, Oxymorphone HCl, Oxytocin,Dinoprostone, Ritodrine, Scopolamine hydrobromide, Sufentanil citrate,and Oxytocic, agents administered to treat depression include serotoninreuptake inhibitors (e.g., Fluoxetine, Escitalopram, Citalopram,Paroxetine, Sertraline, and Venlafaxine); tricyclic antidepressants(e.g., Amitriptyline, Amoxapine, Clomipramine, Desipramine, Dosulepinhydrochloride, Doxepin, Imipramine, Iprindole, Lofepramine,Nortriptyline, Opipramol, Protriptyline, and Trimipramine); monoamineoxidase inhibitors (e.g., Isocarboxazid, Moclobemide, Phenelzine,Tranylcypromine, Selegiline, Rasagiline, Nialamide, Iproniazid,Iproclozide, Toloxatone, Linezolid, Dienolide kavapyronedesmethoxyyangonin, and Dextroamphetamine); psychostimulants (e.g.,Amphetamine, Methamphetamine, Methylphenidate, and Arecoline);antipsychotics (e.g., Butyrophenones, Phenothiazines, Thioxanthenes,Clozapine, Olanzapine, Risperidone, Quetiapine, Ziprasidone,Amisulpride, Paliperidone, Symbyax, Tetrabenazine, and Cannabidiol); andmood stabilizers (e.g., Lithium carbonate, Valproic acid, Divalproexsodium, Sodium valproate, Lamotrigine, Carbamazepine, Gabapentin,Oxcarbazepine, and Topiramate), agents administered to treat anxietyinclude serotonin reuptake inhibitors, mood stabilizers, benzodiazepines(e.g., Alprazolam, Clonazepam, Diazepam, and Lorazepam), tricyclicantidepressants, monoamine oxidase inhibitors, and beta-blockers, andother weight loss agents, including serotonin and noradrenergicre-uptake inhibitors; noradrenergic re-uptake inhibitors; selectiveserotonin re-uptake inhibitors; and intestinal lipase inhibitors.Particular weight loss agents include orlistat, sibutramine,methamphetamine, ionamin, phentermine, bupropion, diethylpropion,phendimetrazine, benzphetermine, and topamax.

In some embodiments, contemplated methods may further comprisingassessing one or more indices of on-going weight loss, e.g. the ketonebody production level in a patient; and optionally adjusting the amountadministered; thereby optimizing the therapeutic efficacy of said MetAP2inhibitor.

Administration and Formulation

Contemplated herein are formulations suitable for non-parenteraladministration of MetAP2 inhibitors. For example, in certainembodiments, a patient may have a lower systemic exposure (e.g. at leastabout 2, 3, 5, 10, 20, or at least about 30% less systemic exposure) tothe non-parenterally administered (e.g. oral administration) of a MetAP2inhibitor as compared to a patient parenterally administered (e.g.subcutaneously) the same dose of the MetAP2 inhibitor. For example,non-parenterally administered (e.g. orally administered) MetAP2inhibitors (e.g. an irreversible inhibitor) may bind less to MetAP2 ascompared to subcutaneously administered MetAP2 inhibitors.

Contemplated non-parenteral administration includes oral, buccal,transdermal (e.g. by a dermal patch), topical, inhalation, or sublingualadministration, or e.g., ocular, pulmonary, nasal, rectal or vaginaladministration.

In another embodiment, provided herein are effective dosages, e.g. adaily dosage of a MetAP2 inhibitor, that may not substantially modulateor suppress angiogenesis. For example, provided here are methods thatinclude administering doses of MetAP2 inhibitors that are effective forweight loss, but are significantly smaller doses than that necessary tomodulate and/or suppress angiogenesis (which may typically require about12.5 mg/kg to about 50 mg/kg or more). For example, contemplated dosageof a MetAP2 inhibitor in the methods described herein may includeadministering about 25 mg/day, about 10 mg/day, about 5 mg/day, about 3mg/day, about 2 mg/day, about 1 mg/day, about 0.75 mg/day, about 0.5mg/day, about 0.1 mg/day, about 0.05 mg/day, or about 0.01 mg/day.

For example, an effective amount of the drug for weight loss in apatient may be about 0.0001 mg/kg to about 25 mg/kg of body weight perday. For example, a contemplated dosage may from about 0.001 to 10 mg/kgof body weight per day, about 0.001 mg/kg to 1 mg/kg of body weight perday, about 0.001 mg/kg to 0.1 mg/kg of body weight per day or about0.005 to about 0.04 mg/kg or about 0.005 to about 0.049 mg/kg of bodyweight a day. In an embodiment a MetAP2 inhibitor such as disclosedherein (e.g. O-(4-dimethlyaminoethoxycinnamoyl)fumagillol), may beadministered about 0.005 to about 0.04 mg/kg of a patient.

For example, provided herein is a method for treating obesity in apatient in need thereof, comprising administering, parenterally (e.g.intravenously) or non-parenterally, about 0.005 to about 0.04 mg/kg of aMetAP2 inhibitor selected fromO-(4-dimethylaminoethoxycinnamoyl)fumagillol and pharmaceuticallyacceptable salts thereof (for example, an oxalate salt), to saidpatient. Such a method, upon administration of said MetAP2 inhibitore.g. daily or weekly, for about 3, 4, 5 or 6 months or more may resultin at least a 10%, 20%, 30%, or 40% or more weight loss based on thepatient's original weight.

Contemplated methods may include administration of a compositioncomprising a MetAP2 inhibitor, for example, hourly, twice hourly, everythree to four hours, daily, twice daily, 1, 2, 3 or 4 times a week,every three to four days, every week, or once every two weeks dependingon half-life and clearance rate of the particular composition orinhibitor.

Treatment can be continued for as long or as short a period as desired.The compositions may be administered on a regimen of, for example, oneto four or more times per day. A suitable treatment period can be, forexample, at least about one week, at least about two weeks, at leastabout one month, at least about six months, at least about 1 year, orindefinitely. A treatment period can terminate when a desired result,for example a weight loss target, is achieved. For example, when aboutloss of about 20% body weight, about 30% body weight or more has beenachieved. A treatment regimen can include a corrective phase, duringwhich a MetAP2 inhibitor dose sufficient to provide reduction of excessadiposity is administered, followed by a maintenance phase, during whicha lower MetAP2 inhibitor dose sufficient to prevent re-development ofexcess adiposity is administered.

For pulmonary (e.g., intrabronchial) administration, MetAP2 inhibitorscan be formulated with conventional excipients to prepare an inhalablecomposition in the form of a fine powder or atomizable liquid. Forocular administration, MetAP2 inhibitors can be formulated withconventional excipients, for example, in the form of eye drops or anocular implant. Among excipients useful in eye drops are viscosifying orgelling agents, to minimize loss by lacrimation through improvedretention in the eye.

Liquid dosage forms for oral or other administration include, but arenot limited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeagent(s), the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the ocular, oral, or other systemically-delivered compositionscan also include adjuvants such as wetting agents, and emulsifying andsuspending agents.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition may include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants, or patches. Theactive agent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. For example, cutaneous routes of administration are achievedwith aqueous drops, a mist, an emulsion, or a cream.

Transdermal patches may have the added advantage of providing controlleddelivery of the active ingredients to the body. Such dosage forms can bemade by dissolving or dispensing the compound in the proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the compound in a polymermatrix or gel.

When administered in lower doses, injectable preparations are alsocontemplated herein, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents.

Compositions for rectal or vaginal administration may be suppositorieswhich can be prepared by mixing a MetAP2 inhibitor with suitablenon-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax which are solid at ambient temperature butliquid at body temperature and therefore melt in the rectum or vaginalcavity and release the active agent(s). Alternatively, contemplatedformulations can be administered by release from a lumen of an endoscopeafter the endoscope has been inserted into a rectum of a subject.

Oral dosage forms, such as capsules, tablets, pills, powders, andgranules, may be prepared using any suitable process known to the art.For example, a MetAP2 inhibitor may be mixed with enteric materials andcompressed into tablets.

Alternatively, formulations of the invention are incorporated intochewable tablets, crushable tablets, tablets that dissolve rapidlywithin the mouth, or mouth wash.

EXAMPLES

The examples which follow are intended in no way to limit the scope ofthis invention but are provided to illustrate aspects of the disclosedmethods. Many other embodiments of this invention will be apparent toone skilled in the art.

Example 1 Fumagillin Increases Levels of Thioredoxin-1 (TXN) in aDose-Dependent Manner

B16F10 cells were treated with increasing concentrations of fumagillin.N-terminal chymotryptic fragments of TXN were measured by liquidchromatography/mass spectrometry (LC/MS). Fumagillin significantlyincreased the cellular level of TXN containing an intact N-terminalmethionine [TXN(1-4)] and caused significant accumulation of N-acetylTXN(1-4), consistent with a role for fumagillin as a MetAP2 inhibitor.See FIGS. 1-2. The EC50 of fumagillin to increase Met-TXN acetylatedMet-TXN levels is <10 nM.

Similar results were seen in Cos-1 and 293T cells that were transfectedwith FLAG-tagged TXN in the absence (TXN) and presence (TXN+F) offumagillin. Fumagillin significantly increased the expression on TXN inboth Cos-1 and 293T cells. See FIG. 3. EC50 of fumagillin to increaseTXN levels is <10 nM. See FIG. 4.

Example 2 Oral Administration of Fumagillin Reduces Body Fat in Mammals

Oral administration of the MetAP2 inhibitor fumagillin causes anincrease in circulating ketone bodies, as measured by concentration ofbeta hydroxybutyrate, which indicates an increase in the breakdown ofstored fat. C57BL/6 mice conditioned on a high fat diet (also referredto as a diet-induced obese diet or DIO) were treated with fumagillin(ZGN-201) administered by oral gavage at doses of 0.3 or 3 mg/kg dailyfor 10 days resulting in an increase in beta hydroxybutyrate (FIG. 5A)concentrations, without an increase in free fatty acid concentration(FIG. 5B) as well as a reduction in body weight by 7 to 15 percent (datanot shown). Increased hydroxybutyrate concentration persisted at longertime points, as was seen in C57BL/6 mice conditioned on a high fat dietand treated with fumagillin (ZGN-201) as a diet admixture at a dose of 1mg/kg daily for 250 days (FIG. 5C).

In addition to showing an increase in beta hydroxybutyrateconcentration, C57BL/6 mice conditioned on a high fat diet and treatedwith fumagillin (ZGN-201) as a diet admixture (1 mg/kg daily for 250days) also experienced a reduction in body fat by approximately 40percent (FIG. 6). Once attaining the maximum weight reduction, micetreated daily with 1 mg/kg fumagillin maintained a weight that was 25%less than that of age-matched mice maintained on normal mouse chow diet(lean mice, FIG. 6) for the duration of the experiment. The size andappearance of epididymal fat, peri-renal fat and liver infumagillin-treated mice was more similar to that of age-matched leanmice than that of untreated mice on a high fat diet (FIG. 7).Retroperitoneal fat pad weight in fumagillin-treated mice was also moresimilar to that of age-matched lean mice than that of untreated mice ona high fat diet (FIG. 8).

Adipose tissue in mice fed a high-fat diet show distinctive crown-likestructures not seen in lean mice. These crown-like structures were alsoabsent from the adipose tissue of fumagillin-treated mice. Additionally,adipocyte size in fumagillin-treated mice was more similar to that ofage-matched lean mice than that of untreated mice on a high fat diet.

Fasted glucose and insulin levels, as well as insulin-glucose product,in fumagillin-treated mice was more similar to that of age-matched leanmice than that of untreated mice on a high fat diet (FIG. 9), indicatingthat fumagillin normalizes sensitivity to insulin in mice on a high fatdiet.

Example 3 Fumagillin Reduces Food Intake During the Peak of Weight Loss

High fat diet-fed C57BL/6 mice were treated with 1 mg/kg of the MetAP2inhibitor fumagillin (ZGN-201, squares) or vehicle (diamonds) for 14days (FIG. 10). Body weights (left panel) were determined daily, whilefood intake (right panel) was assessed every other day by measuring thefood consumed by each cage of three mice over the two day period. Datashown in FIG. 10 are means±SEM, n=9 animals. As illustrated in FIG. 10,weight loss during treatment with MetAP2 inhibitors follows a sequenceof three distinct phases. First, during the initial two to three days oftreatment, no overt changes in food intake or body weight are observed.During this time period key changes in MetAP2 substrates occur,including glyceraldehyde-3-phosphate dehydrogenase and thioredoxin (datanot shown). Second, the animals enter a period of rapid weight loss thatproceeds until essentially all excess fat deposits are consumed. Duringthis second period, fat oxidation is enhanced and food intake isinhibited.

Weight loss following treatment with therapeutically relevant doses ofMetAP2 inhibitors stops once animals attain a normal body composition.Once weight loss reaches a nadir, fumagillin-treated animals enter athird phase in which their food intake returns to normal levels despitemaintaining a normal body weight and improved metabolic controlevidenced by reduced insulin and glucose concentrations. This thirdphase can be maintained indefinitely provided the drug is continued (seeFIG. 10), or presumably can be maintained in practice through othercomplementary pharmacologic, dietary, or lifestyle interventions.

Example 4 Oral Administration of Fumagillin Results in Fat Loss withMinimal Change in Lean Tissue

C57BL/6 mice were conditioned on a low-fat diet (LF), a high fat diet(HF), or a high-fat diet and treated with fumagillin (ZGN-201)administered by oral gavage at doses of 0.1 or 0.3 mg/kg for 28 days.Either dose of fumagillin caused a reduction in body weight down to thelevel of low fat fed mice. To determine whether the weight loss resultedfrom loss of fat or loss of lean tissue, the percentage change in fattissue and lean tissue was determined for LF mice, HF mice, HF micetreated with 0.1 mg/kg fumagillin, and HF mice treated with 0.3 mg/kgfumagillin. As shown in FIG. 11, HF mice treated with either dose offumagillin experienced a 40-50% reduction in fat (compared to anincrease in fat in both the LF and HF control mice), but only a 5-10%reduction in lean tissue, indicating that fumagillin-induced weight lossresults primarily from loss of fat, rather than loss of lean tissue.

Upon histological examination, the studies of treated obese mice show noeffect on growth of new blood vessels in growing fat depots and noeffect on blood vessel size or density when compared to animals in whichweight loss has been induced by simple energy (food) restriction.

Example 5 Orally Administered Compounds Having MetAP-2 Inhibitory CoresCause Weight Loss in Diet-Induced Obese Mice

A weight loss study was conducted in obese mice. The mice in this studywere not genetically obese, but prior to and during the study, obesitywas induced by a high-fat diet. Twelve week-old C57BL/6NTac mice,maintained on a 60% fat diet prior to and during the study, wereseparated into seven groups, eight animals per group. Average bodyweight of the mice was approximately 47 g at the start of the study.

Each of six groups was orally administered 1.0 mg/kg of a compound(fumagillin and compounds B. C, D, E, F and G as disclosed herein), in10% gelucire in deoinised water. Each of these six groups wasadministered a different compound. One group was orally administeredfumagillin at 1.0 mg/kg (group 201) in 10% gelucire in deoinised water,and one group was administered 10% gelucire in deoinised water(vehicle). Mice received administrations once a day for 7 days.

Data show that mice administered fumagillin lost the most weight overthe course of the 8 days (FIG. 13). Mice in groups B, C, E, and E alsolost weight over the course of the 8 days, with mice in group 233 losingthe most weight of these four groups (FIG. 13). (Data analyzed by ANCOVAwith body weight on Day 1 as covariate followed by multiple testsagainst vehicle group: *p<0.05; **p<0.01, ***p<0.001.)

Example 6

A weight loss study was conducted in obese mice, similar to Example 5.Twelve week-old C57BL/6NTac mice, maintained on a 60% fat diet prior toand during the study, were separated into seven groups, eight animalsper group. Average body weight of the mice was approximately 47 g at thestart of the study.

The group was orally administered 100.0 mg/kg of a compound H, in 10%gelucire in deoinised water. Mice received administrations once a dayfor 7 days. After 7 days, the average weight loss of the mice was 13.2%

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

1. A method for treating obesity in a patient in need thereof, whereinsaid patient has a BMI of 30 kg/m² or greater, or has a BMI of 25 kg/m²to 29.9 kg/m² and an obesity related co-morbidity, comprisingsubcutaneously administering, every three to four days, a dose of about0.005 to about 0.049 mg/kg of a MetAP2 inhibitor selected fromO-(4-dimethylaminoethoxycinnamoyl) fumagillol and pharmaceuticallyacceptable salts thereof, to said patient.
 2. A method of treatingobesity in a patient having a BMI of 30 kg/m² or greater, comprisingparenterally administering 2 or 3 times a week to said patient a dose ofabout 0.005 to about 0.049 mg/kg of a compound selected from the groupconsisting of O-(4-dimethylaminoethoxycinnamoyl) fumagillol and apharmaceutically acceptable salt thereof.